Identification of Structural Determinants of the Transport of the Dehydroascorbic Acid Mediated by Glucose Transport GLUT1.

GLUT1 is a facilitative glucose transporter that can transport oxidized vitamin C (i.e., dehydroascorbic acid) and complements the action of reduced vitamin C transporters. To identify the residues involved in human GLUT1's transport of dehydroascorbic acid, we performed docking studies in the 5 Å grid of the glucose-binding cavity of GLUT1. The interactions of the bicyclic hemiacetal form of dehydroascorbic acid with GLUT1 through hydrogen bonds with the -OH group of C3 and C5 were less favorable than the interactions with the sugars transported by GLUT1. The eight most relevant residues in such interactions (i.e., F26, Q161, I164, Q282, Y292, and W412) were mutated to alanine to perform functional studies for dehydroascorbic acid and the glucose analog, 2-deoxiglucose, in Xenopus laevis oocytes. All the mutants decreased the uptake of both substrates to less than 50%. The partial effect of the N317A mutant in transporting dehydroascorbic acid was associated with a 30% decrease in the Vmax compared to the wildtype GLUT1. The results show that both substrates share the eight residues studied in GLUT1, albeit with a differential contribution of N317. Our work, combining docking with functional studies, marks the first to identify structural determinants of oxidized vitamin C's transport via GLUT1.

The activity of IKCa and BKCa channels contributes to insulin-mediated NO synthesis and vascular tone regulation in human umbilical vein.

Insulin regulates the l-arginine/nitric oxide (NO) pathway in human umbilical vein endothelial cells (HUVECs), increasing the plasma membrane expression of the l-arginine transporter hCAT-1 and inducing vasodilation in umbilical and placental veins. Placental vascular relaxation induced by insulin is dependent of large conductance calcium-activated potassium channels (BKCa), but the role of KCa channels on l-arginine transport and NO synthesis is still unknown. The aim of this study was to determine the contribution of KCa channels in both insulin-induced l-arginine transport and NO synthesis, and its relationship with placental vascular relaxation. HUVECs, human placental vein endothelial cells (HPVECs) and placental veins were freshly isolated from umbilical cords and placenta from normal pregnancies. Cells or tissue were incubated in absence or presence of insulin and/or tetraethylammonium, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, iberiotoxin or NG-nitro-l-arginine methyl ester. l-Arginine uptake, plasma membrane polarity, NO levels, hCAT-1 expression and placenta vascular reactivity were analyzed. The inhibition of intermediate-conductance KCa (IKCa) and BKCa increases l-arginine uptake, which was related with protein abundance of hCAT-1 in HUVECs. IKCa and BKCa activities contribute to NO-synthesis induced by insulin but are not directly involved in insulin-stimulated l-arginine uptake. Long term incubation (8 h) with insulin increases the plasma membrane hyperpolarization and hCAT-1 expression in HUVECs and HPVECs. Insulin-induced relaxation in placental vasculature was reversed by KCa inhibition. The results show that the activity of IKCa and BKCa channels are relevant for both physiological regulations of NO synthesis and vascular tone regulation in the human placenta, acting as a part of negative feedback mechanism for autoregulation of l-arginine transport in HUVECs.

Expression and purification of the surface proteins from Andes virus.

Andes virus is the main causative agent of Hantavirus cardiopulmonary syndrome in South America. There are currently no vaccines or treatments against Andes virus. However, there are several evidences suggesting that antibodies against Andes virus envelope glycoproteins may be enough to confer full protection against Hantavirus cardiopulmonary syndrome. The goal of the present work was to express, purify and characterize the extracellular domains of Andes virus glycoproteins Gn and Gc. We generated two adenoviral vectors encoding the extracellular domains of Andes virus glycoproteins Gn and Gc. Both molecules were expressed by adenoviral transduction in SiHa cells. We found that sGc ectodomain was mainly secreted into the culture medium, whereas sGn was predominantly retained inside the cells. Both molecules were expressed at very low concentrations (below 1 µg/mL). Treatment with the proteasome inhibitor ALLN raised sGc concentration in the cell culture medium, but did not affect expression levels of sGn. Both ectodomains were purified by immobilized metal ion affinity chromatography, and were recognized by sera from persons previously exposed to Andes virus. To our knowledge, this is the first work that addresses the expression and purification of Andes virus glycoproteins Gn and Gc. Our results demonstrate that sGn and sGc maintain epitopes that are exposed on the surface of the viral envelope. However, our work also highlights the need to explore new strategies to achieve high-level expression of these proteins for development of a vaccine candidate against Andes virus.

Bone stroma-derived cells change coregulators recruitment to androgen receptor and decrease cell proliferation in androgen-sensitive and castration-resistant prostate cancer cells.

Prostate cancer (CaP) bone metastasis is an early event that remains inactive until later-stage progression. Reduced levels of circulating androgens, due to andropause or androgen deprivation therapies, alter androgen receptor (AR) coactivator expression. Coactivators shift the balance towards enhanced AR-mediated gene transcription that promotes progression to androgen-resistance. Disruptions in coregulators may represent a molecular switch that reactivates latent bone metastasis. Changes in AR-mediated transcription in androgen-sensitive LNCaP and androgen-resistant C4-2 cells were analyzed for AR coregulator recruitment in co-culture with Saos-2 and THP-1. The Saos-2 cell line derived from human osteosarcoma and THP-1 cell line representing human monocytes were used to display osteoblast and osteoclast activity. Increased AR activity in androgen-resistant C4-2 was due to increased AR expression and SRC1/TIF2 recruitment and decreased SMRT/NCoR expression. AR activity in both cell types was decreased over 90% when co-cultured with Saos-2 or THP-1 due to dissociation of AR from the SRC1/TIF2 and SMRT/NCoR coregulators complex, in a ligand-dependent and cell-type specific manner. In the absence of androgens, Saos-2 decreased while THP-1 increased proliferation of LNCaP cells. In contrast, both Saos-2 and THP-1 decreased proliferation of C4-2 in absence and presence of androgens. Global changes in gene expression from both CaP cell lines identified potential cell cycle and androgen regulated genes as mechanisms for changes in cell proliferation and AR-mediated transactivation in the context of bone marrow stroma cells.

Resolution of the direct interaction with and inhibition of the human GLUT1 hexose transporter by resveratrol from its effect on glucose accumulation.

Resveratrol acts as a chemopreventive agent for cancer and as a potential antiobesity and antidiabetic compound, by leading to reduced body fat and improved glucose homeostasis. The exact mechanisms involved in improving hyperglycemic state are not known, but most of the glucose uptake into mammalian cells is facilitated by the GLUT hexose transporters. Resveratrol is structurally similar to isoflavones such as genistein, which inhibit the glucose uptake facilitated by the GLUT1 hexose transporter. Here we examined the direct effects of resveratrol on glucose uptake and accumulation in HL-60 and U-937 leukemic cell lines, which express mainly GLUT1, under conditions that discriminate transport from the intracellular substrate phosphorylation/accumulation. Resveratrol blocks GLUT1-mediated hexose uptake and thereby affects substrate accumulation on these cells. Consequently, we characterized the mechanism involved in inhibition of glucose uptake in human red cells. Resveratrol inhibits glucose exit in human red cells, and the displacement of previously bound cytochalasin B revealed the direct interaction of resveratrol with GLUT1. Resveratrol behaves as a competitive blocker of glucose uptake under zero-trans exit and exchange kinetic assays, but it becomes a mixed noncompetitive blocker when zero-trans entry transport was assayed, suggesting that the binding site for resveratrol lies on the endofacial face of the transporter. We propose that resveratrol interacts directly with the human GLUT1 hexose transporter by binding to an endofacial site and that this interaction inhibits the transport of hexoses across the plasma membrane. This inhibition is distinct from the effect of resveratrol on the intracellular phosphorylation/accumulation of glucose.

Co-Formulation of Recombinant Porcine IL-18 Enhances the Onset of Immune Response in a New Lawsonia intracellularis Vaccine.

Pig is one of the most consumed meats worldwide. One of the main conditions for pig production is Porcine Enteropathy caused by Lawsonia intracellularis. Among the effects of this disease is chronic mild diarrhea, which affects the weight gain of pigs, generating economic losses. Vaccines available to prevent this condition do not have the desired effect, but this limitation can be overcome using adjuvants. Pro-inflammatory cytokines, such as interleukin 18 (IL-18), can improve an immune response, reducing the immune window of protection. In this study, recombinant porcine IL-18 was produced and expressed in Escherichia coli and Pichia pastoris. The protein's biological activity was assessed in vitro and in vivo, and we determined that the P. pastoris protein had better immunostimulatory activity. A vaccine candidate against L. intracellularis, formulated with and without IL-18, was used to determine the pigs' cellular and humoral immune responses. Animals injected with the candidate vaccine co-formulated with IL-18 showed a significant increase of Th1 immune response markers and an earlier increase of antibodies than those vaccinated without the cytokine. This suggests that IL-18 acts as an immunostimulant and vaccine adjuvant to boost the immune response against the antigens, reducing the therapeutic window of recombinant protein-based vaccines.

Noncompetitive blocking of human GLUT1 hexose transporter by methylxanthines reveals an exofacial regulatory binding site.

Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2-3 mM) in exchange and zero-trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero-trans influx assays (inhibition constant values of 5-12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.

Hexose transporter GLUT1 harbors several distinct regulatory binding sites for flavones and tyrphostins.

The facilitative hexose transporter GLUT1 activity is blocked by tyrosine kinase inhibitors that include natural products such as flavones and isoflavones and synthetic compounds such as tyrphostins, molecules that are structurally unrelated to the transported substrates [Vera, et al. (2001) Biochemistry, 40, 777-790]. Here we analyzed the interaction of GLUT1 with quercetin (a flavone), genistein (an isoflavone), and tyrphostin A47 and B46 to evaluate if they share one common or have several binding sites on the protein. Kinetic assays showed that genistein, quercetin, and tyrphostin B46 behave as competitive inhibitors of equilibrium exchange and zero-trans uptake transport and noncompetitive inhibitors of net sugar exit out of human red cells, suggesting that they interact with the external surface of the GLUT1 molecule. In contrast, tyrphostin A47 was a competitive inhibitor of equilibrium exchange and zero-trans exit transport and a noncompetitive inhibitor of net sugar entry into red cells, suggesting that it interacts with the cytoplasmic surface of the transporter. Genistein protected GLUT1 against iodide-elicited fluorescence quenching and also decreased the affinity of d-glucose for its external binding site, while quercetin and tyrphostins B46 and A47 promoted fluorescence quenching and did not affect the external d-glucose binding site. These findings are explained by a carrier that presents at least three binding sites for tyrosine kinase inhibitors, in which (i) genistein interacts with the transporter in a conformation that binds glucose on the external surface (outward-facing conformation), in a site which overlaps with the external binding site for d-glucose, (ii) quercetin and tyrphostin B46 interact with the GLUT1 conformation which binds glucose by the internal side of the membrane (inward-facing conformation), but to a site accessible from the external surface of the protein, and (iii) the binding site for tyrphostin A47 is accessible from the inner surface of GLUT1 by binding to the inward-facing conformation of the transporter. These data provide groundwork for a molecular understanding of how the tyrosine kinase inhibitors directly affect glucose transport in animal cells.

Histidine residues in the Na+-coupled ascorbic acid transporter-2 (SVCT2) are central regulators of SVCT2 function, modulating pH sensitivity, transporter kinetics, Na+ cooperativity, conformational stability, and subcellular localization.

Na(+)-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His(109), His(203), His(206), His(269), and His(413), are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na(+) cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His(413), localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na(+) and loss of Na(+) cooperativity, which leads to a decreased V(max) without altering the transport K(m); (ii) exofacial histidine residues His(203), His(206), and His(413) may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K(m); and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.

Transport of Vitamin C in Cancer.

Significance: Vitamin C is a powerful antioxidant that has an intricate relationship with cancer and has been studied for more than 60 years. However, the specific mechanisms that allow malignant cells to uptake, metabolize, and compartmentalize vitamin C remain unclear. In normal human cells, two different transporter systems are responsible for its acquisition: glucose transporters (GLUTs) transport the oxidized form of vitamin C (dehydroascorbic acid) and sodium-coupled ascorbic acid transporters (SVCTs) transport the reduced form (ascorbic acid [AA]). In this study, we review the mechanisms described for vitamin C uptake and metabolization in cancer. Recent Advances: Several studies performed recently in vivo and in vitro have provided the scientific community a better understanding of the differential capacities of cancer cells to acquire vitamin C: tumors from different origins do not express SVCTs in the plasma membrane and are only able to acquire vitamin C in its oxidized form. Interestingly, cancer cells differentially express a mitochondrial form of SVCT2. Critical Issues: Why tumors have reduced AA uptake capacity at the plasma membrane, but develop the capacity of AA transport within mitochondria, remains a mystery. However, it shows that understanding vitamin C physiology in tumor survival might be key to decipher the controversies in its relationship with cancer. Future Directions: A comprehensive analysis of the mechanisms by which cancer cells acquire, compartmentalize, and use vitamin C will allow the design of new therapeutic approaches in human cancer. Antioxid. Redox Signal. 35, 61-74.

PrP C as a Transducer of Physiological and Pathological Signals.

After the discovery of prion phenomenon, the physiological role of the cellular prion protein (PrP C ) remained elusive. In the past decades, molecular and cellular analysis has shed some light regarding interactions and functions of PrP C in health and disease. PrP C , which is located mainly at the plasma membrane of neuronal cells attached by a glycosylphosphatidylinositol (GPI) anchor, can act as a receptor or transducer from external signaling. Although the precise role of PrP C remains elusive, a variety of functions have been proposed for this protein, namely, neuronal excitability and viability. Although many issues must be solved to clearly define the role of PrP C , its connection to the central nervous system (CNS) and to several misfolding-associated diseases makes PrP C an interesting pharmacological target. In a physiological context, several reports have proposed that PrP C modulates synaptic transmission, interacting with various proteins, namely, ion pumps, channels, and metabotropic receptors. PrP C has also been implicated in the pathophysiological cell signaling induced by ß-amyloid peptide that leads to synaptic dysfunction in the context of Alzheimer's disease (AD), as a mediator of Aß-induced cell toxicity. Additionally, it has been implicated in other proteinopathies as well. In this review, we aimed to analyze the role of PrP C as a transducer of physiological and pathological signaling.

Therapeutic Use of Vitamin C in Cancer: Physiological Considerations.

Since the early studies of William J. McCormick in the 1950s, vitamin C has been proposed as a candidate for the treatment of cancer. A number of reports have shown that pharmacological concentrations of vitamin C selectively kill cancer cells in vitro and decrease the growth rates of a number of human tumor xenografts in immunodeficient mice. However, up to the date there is still doubt regarding this possible therapeutic role of vitamin C in cancer, mainly because high dose administration in cancer patients has not showed a clear antitumor activity. These apparent controversial findings highlight the fact that we lack information on the interactions that occurs between cancer cells and vitamin C, and if these transformed cells can uptake, metabolize and compartmentalize vitamin C like normal human cells do. The role of SVCTs and GLUTs transporters, which uptake the reduced form and the oxidized form of vitamin C, respectively, has been recently highlighted in the context of cancer showing that the relationship between vitamin C and cancer might be more complex than previously thought. In this review, we analyze the state of art of the effect of vitamin C on cancer cells in vitro and in vivo, and relate it to the capacity of cancer cells in acquiring, metabolize and compartmentalize this nutrient, with its implications on the potential therapeutic role of vitamin C in cancer.

Endofacial competitive inhibition of the glucose transporter 1 activity by gossypol.

Gossypol is a natural disesquiterpene that blocks the activity of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, Chinese hamster ovary cells overexpressing GLUT1, and human erythrocytes, gossypol inhibited hexose transport in a concentration-dependent fashion, indicating that blocking of GLUT1 activity is independent of cellular context. With the exception of red blood cells, the inhibition of cellular transport was instantaneous. Gossypol effect was specific for the GLUT1 transporter since it did not alter the uptake of nicotinamide by human erythrocytes. Gossypol affects the glucose-displaceable binding of cytochalasin B to GLUT1 in human erythrocyte ghost in a mixed noncompetitive way, with a K(i) value of 20 microM. Likewise, GLUT1 fluorescence was quenched approximately 80% by gossypol, while Stern-Volmer plots for quenching by iodide displayed increased slopes by gossypol addition. These effects on protein fluorescence were saturable and unaffected by the presence of D-glucose. Gossypol did not alter the affinity of D-glucose for the external substrate site on GLUT1. Kinetic analysis of transport revealed that gossypol behaves as a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but it acts as a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport, which is consistent with interaction at the endofacial surface, but not at the exofacial surface of the transporter. Thus, gossypol behaves as a quasi-competitive inhibitor of GLUT1 transport activity by binding to a site accessible through the internal face of the transporter, but it does not, in fact, compete with cytochalasin B binding. Our observations suggest that some effects of gossypol on cellular physiology may be related to its ability to disrupt the normal hexose flux through GLUT1, a transporter expressed in almost every kind of mammalian cell and responsible for the basal uptake of glucose.

GLUT1 and GLUT8 support lactose synthesis in Golgi of murine mammary epithelial cells.

The mammary gland increases energy requirements during pregnancy and lactation to support epithelial proliferation and milk nutrients synthesis. Lactose, the principal carbohydrate of the milk, is synthetized in the Golgi of mammary epithelial cells by lactose synthase from glucose and UPD galactose. We studied the temporal changes in the expression of GLUT1 and GLUT8 in mammary gland and their association with lactose synthesis and proliferation in BALB/c mice. Six groups were used: virgin, pregnant at 2 and 17 days, lactating at 2 and 10 days, and weaning at 2 days. Temporal expression of GLUT1 and GLUT8 transporters by qPCR, western blot and immunohistochemistry, and its association with lactalbumin, Ki67, and cytokeratin 18 within mammary tissue was studied, along with subcellular localization. GLUT1 and GLUT8 transporters increased their expression during mammary gland progression, reaching 20-fold increasing in GLUT1 mRNA at lactation (p < 0.05) and 2-fold at protein level for GLUT1 and GLUT8 (p < 0.05 and 0.01, respectively). The temporal expression pattern was shared with cytokeratin 18 and Ki67 (p < 0.01). Endogenous GLUT8 partially co-localized with 58 K protein and α-lactalbumin in mammary tissue and with Golgi membrane-associated protein 130 in isolated epithelial cells. The spatial-temporal synchrony between expression of GLUT8/GLUT1 and alveolar cell proliferation, and its localization in cis-Golgi associated to lactose synthase complex, suggest that both transporters are involved in glucose uptake into this organelle, supporting lactose synthesis.

Data on SVCT2 transporter expression and localization in cancer cell lines and tissues.

The data presented in this article are related to the research paper entitled "Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer", available in Free Radical Biology and Medicine Journal [1]. In this article, we examined the SVCT2 transporter expression in various breast cancer cell lines using RT-PCR and Western blot assays. In addition, we analyzed the subcellular localization of SVCT2 by immunofluorescence colocalization assays and cellular fractionation experiments. Finally, an analysis of different cancer tissue microarrays immunostained for SVCT2 and imaged by The Human Protein Atlas (https://www.proteinatlas.org) is presented.

Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer.

The potential role of vitamin C in cancer prevention and treatment remains controversial. While normal human cells obtain vitamin C as ascorbic acid, the prevalent form of vitamin C in vivo, the uptake mechanisms by which cancer cells acquire vitamin C has remained unclear. The aim of this study is to characterize how breast cancer cells acquire vitamin C. For this, we determined the expression of vitamin C transporters in normal and breast cancer tissue samples, and in ZR-75, MCF-7, MDA-231 and MDA-468 breast cancer cell lines. At the same time, reduced (AA) and oxidized (DHA) forms of vitamin C uptake experiments were performed in all cell lines. We show here that human breast cancer tissues differentially express a form of SVCT2 transporter, that is systematically absent in normal breast tissues and it is increased in breast tumors. In fact, estrogen receptor negative breast cancer tissue, exhibit the most elevated SVCT2 expression levels. Despite this, our analysis in breast cancer cell lines showed that these cells are not able to uptake ascorbic acid and depend on glucose transporter for the acquisition of vitamin C by a bystander effect. This is consistent with our observations that this form of SVCT2 is completely absent from the plasma membrane and is overexpressed in mitochondria of breast cancer cells, where it mediates ascorbic acid transport. This work shows that breast cancer cells acquire vitamin C in its oxidized form and are capable of accumulated high concentrations of the reduced form. Augmented expression of an SVCT2 mitochondrial form appears to be a common hallmark across all human cancers and might have implications in cancer cells survival capacity against pro-oxidant environments.

Molecular identification and functional characterization of the vitamin C transporters expressed by Sertoli cells.

Vitamin C is an essential micronutrient for the development of male germ cells. In the gonad, the germ cells are isolated from the systemic circulation by the blood-testis barrier, which consists of a basal layer of Sertoli cells that communicate through an extensive array of tight junction complexes. To study the behavior of Sertoli cells as a first approach to the molecular and functional characterization of the vitamin C transporters in this barrier, we used the 42GPA9 cell line immortalized from mouse Sertoli cells. To date, there is no available information on the mechanism of vitamin C transport across the blood-testis barrier. This work describe the molecular identity of the transporters involved in vitamin C transport in these cells, which we hope will improve our understanding of how germ cells obtain vitamin C, transported from the plasma into the adluminal compartment of the seminiferous tubules. RT-PCR analyses revealed that 42GPA9 cells express both vitamin C transport systems, a finding that was confirmed by immunocytochemical and immunoblotting analysis. The kinetic assays using radioactive vitamin C revealed that both ascorbic acid (AA) transporters, SVCT1 and SVCT2, are functionally active. Moreover, the kinetic characteristics of dehydroascorbic acid (DHA) and 3-methylglucose (OMG) transport by 42GPA9 Sertoli cells correspond to facilitative hexose transporters GLUT1, GLUT2 and GLUT3 expressed in these cells. This data is consistent with the concept that Sertoli cells have the ability to take up vitamin C. It is an important finding and contributes to our knowledge of the physiology of male germ cells.

Sustained blockade of ascorbic acid transport associated with marked SVCT1 loss in rat hepatocytes containing increased ascorbic acid levels after partial hepatectomy.

The liver has an extraordinary regenerative capacity in response to partial hepatectomy (PHx), which develops with neither tissue inflammation response nor alterations in the whole organism. This process is highly coordinated and it has been associated with changes in glutathione (GSH) metabolism. However, there are no reports indicating ascorbic acid (AA) levels after partial hepatectomy. AA and GSH act integrally as an antioxidant system that protects cells and tissues from oxidative damage and imbalance observed in a variety of diseases that affect the liver. Although rat hepatocytes are able to synthesize AA and GSH, which are the providers of AA for the whole organism, they also acquire AA from extracellular sources through the sodium-coupled ascorbic acid transporter-1 (SVCT1). Here, we show that hepatocytes from rat livers subjected to PHx increase their GSH and AA levels from 1 to 7 days post hepatectomy, whose peaks precede the peak in cell proliferation observed at 3 days post-hepatectomy. The increase in both antioxidants was associated with higher expression of the enzymes involved in their synthesis, such as the modifier subunit of enzyme glutamine cysteine ligase (GCLM), glutathione synthetase (GS), gulonolactonase (GLN) and gulonolactone oxidase (GULO). Importantly, rat hepatocytes, that normally exhibit kinetic evidence indicating only SVCT1-mediated transport of AA, lost more than 90% of their capacity to transport it at day 1 after PHx without evidence of recovery at day 7. This observation was in agreement with loss of SVCT1 protein expression, which was undetectable in hepatocytes as early as 2h after PHx, with partial recovery at day 7, when the regenerated liver weight returns to normal. We conclude that after PHx, rat hepatocytes enhance their antioxidant capacity by increasing GSH and AA levels prior to the proliferative peak. GSH and AA are increased by de novo synthesis, however paradoxically hepatocytes from rat subjected to PHx also suppress their capacity to acquire AA from extracellular sources through SVCT1.

Insulin Induces Relaxation and Decreases Hydrogen Peroxide-Induced Vasoconstriction in Human Placental Vascular Bed in a Mechanism Mediated by Calcium-Activated Potassium Channels and L-Arginine/Nitric Oxide Pathways.

HIGHLIGHTS Short-term incubation with insulin increases the L-arginine transport in HUVECs.Short-term incubation with insulin increases the NO synthesis in HUVECs.Insulin induces relaxation in human placental vascular bed.Insulin attenuates the constriction induced by hydrogen peroxide in human placenta.The relaxation induced by insulin is dependent on BKCa channels activity in human placenta. Insulin induces relaxation in umbilical veins, increasing the expression of human amino acid transporter 1 (hCAT-1) and nitric oxide synthesis (NO) in human umbilical vein endothelial cells (HUVECs). Short-term effects of insulin on vasculature have been reported in healthy subjects and cell cultures; however, its mechanisms remain unknown. The aim of this study was to characterize the effect of acute incubation with insulin on the regulation of vascular tone of placental vasculature. HUVECs and chorionic vein rings were isolated from normal pregnancies. The effect of insulin on NO synthesis, L-arginine transport, and hCAT-1 abundance was measured in HUVECs. Isometric tension induced by U46619 (thromboxane A2 analog) or hydrogen peroxide (H2O2) were measured in vessels previously incubated 30 min with insulin and/or the following pharmacological inhibitors: tetraethylammonium (KCa channels), iberiotoxin (BKCa channels), genistein (tyrosine kinases), and wortmannin (phosphatidylinositol 3-kinase). Insulin increases L-arginine transport and NO synthesis in HUVECs. In the placenta, this hormone caused relaxation of the chorionic vein, and reduced perfusion pressure in placental cotyledons. In vessels pre-incubated with insulin, the constriction evoked by H2O2 and U46619 was attenuated and the effect on H2O2-induced constriction was blocked with tetraethylammonium and iberiotoxin, but not with genistein, or wortmannin. Insulin rapidly dilates the placental vasculature through a mechanism involving activity of BKCa channels and L-arginine/NO pathway in endothelial cells. This phenomenon is related to quick increases of hCAT-1 abundance and higher capacity of endothelial cells to take up L-arginine and generate NO.

Differential signalling for enhanced hexose uptake by interleukin (IL)-3 and IL-5 in male germ cells.

We studied the expression and function of the IL (interleukin)-3 and IL-5 family of receptors in male germ cells. RT (reverse transcription)-PCR showed expression of mRNAs encoding the alpha and beta subunits of the IL-3 and IL-5 receptors in human testis, and the presence of IL-3 and IL-5 receptors alpha and beta proteins was confirmed by immunoblotting with anti-alpha and anti-beta antibodies. The immunolocalization studies showed expression of these receptors in the germ line in the human testis and in human and bovine ejaculated spermatozoa. Functional studies with bull spermatozoa indicated that IL-3 signalled for increased uptake of hexoses in these cells at picomolar concentrations compatible with expression of functional high-affinity IL-3 receptors in these cells. In contrast, IL-5 failed to induce increased hexose uptake in bull spermatozoa. Experiments using HL-60 eosinophils that express functional IL-3 and IL-5 receptors confirmed that IL-3, but not IL-5, signalled for increased hexose uptake. Our findings suggest that differential signalling for increased hexose uptake by heteromeric high-affinity IL-3 and IL-5 receptors in mammalian spermatozoa is a property that depends on the identity of the alpha-subunit forming part of the alphabeta-complex and is not a property specific to the germ cells.